The invention concerns a solid-state fermenter in particular for high volumes as well as a procedure for solid-state fermentation.
The submerged or solid-state fermentation is used for the mass cultivation of microorganisms with the goal of either isolating the microorganisms themselves or the metabolic product or a microbial altered substrate (e.g. in the food-processing industry). Whereas nowadays submerged fermenters (fermenters with a liquid nutritive substrate) already are built with a capacity of up to 200.000 liters, it has still not yet been achieved to build solid state fermenters (fermenter with a solid nutritive substrate) with economically relevant volumes, that can be kept free of contaminations by foreign micro-organisms for longer periods and that allow an optimum in cultivation care at the same time. However, certain filamentous fungi need surface structures, which allow them to develop and sporulate there. The largest fermenter for the production of filamentous fungi, which avoids any foreign contamination is located in the INRA in France (Durand 1997, verbal communication) and has a capacity of 50 liters. However, the capacity of this fermenter is not at all sufficient for an economic production of fungal spores that can be used, e.g., as biological agricultural pesticides.
The solid-state fermentation (SSF) is defined as growth of microorganismsxe2x80x94usually fungixe2x80x94on solid substrates in a defined gas phase, but without a free water phase. SSF was already used for the production of fermented food, of enzyme products (Koji) or of edible mushrooms in certain territories of the Orient, Asia and Africa in the Ancient World. The efforts in the Western countries were focused on the submerged fermentation since 1940; whereas the SSF was only used for a reprocessing of organic waste. However, many institutes and firms recently show their interest in the SSF, because there are certain advantages compared to the submerged fermentation. Such advantages compared to the submerged fermentation are:
Possibility of an effective production of secondary metabolites such as enzymes, aroma substances, aromatic substances and coloring substances as well as pharmaceutically active substances
Possibility of a production of microorganisms as biological agents in agricultural pesticides
Elimination of toxins or other detrimental substances from food and feeding stuff or enrichment of proteins or vitamins this stuff.
Fundamentally, there are 6 types of solid-state fermenters:
1. tray bioreactor
2. packed bed bioreactor
3. rotary drum bioreactor
4. swing solid state bioreactor
5. stirred vessel bioreactor
6. air solid fluidized bed bioreactor
The first typexe2x80x94the tray bioreactorxe2x80x94, where the substrate to be fermented is spread out flatly in a container especially intended for this purpose and that is incubated in a room, which is especially air-conditioned for this reason (xe2x80x98Kojixe2x80x99xe2x80x94Raum, Ramana Murthy, M. V.; Karanth, N. G.; Raghava Rao, K. S. M. S.: Advance in Applied Microbiology 38 (1993), 99-147), can be used for the production of large amounts of the product, however, it has to be possible to neglect a small contamination by nucleus of crystallization by this method. Moreover, reactor and method are very space- and labor-intensive. The fermented substrate has to be moved manually within the containers. It is not appropriate for the production of large amounts of fungal spores of little competitive species.
In the xe2x80x98packed bed bioreactorxe2x80x99 a moist granular substrate, which is located in a closed container, is inoculated with a micro-organism, which develops in there without the substrate being moved. For that purpose, the substrate has to be perfused constantly by air. The following problems occur, that do not allow the use of large amounts of substrate from the beginning.
1. The micro-organism produces heat (300 kJ per kg dry weight and hour, Saucedo-Castaneda, G.; Gutierrez-Rojas, M.; Bacquet, G.; Raimbault, M.; Viniegra-Gonzalez, G.: Biotechnologie and Bioengeniering 35 (1990), 802-808), which can either be evacuated through the outer wall of the container or through an increased air-circulation (evaporation coldness). This is not possible, if the containers have large volumes. The micro-organisms slow down their growth with an increase in heat evolution and finally necrotize.
2. A constant aeration dries the substrate out. Thus, the xe2x80x98lossxe2x80x99 caused by this, creates air-channels. Their existence cannot guarantee an even aeration of the substrate any longer. The gradual drying out of the substrate also leads to deterioration in growth of the micro-organism.
The xe2x80x98rotary drum bioreactorxe2x80x99 consists of a cylindrical container, which is allocated horizontally and pivoted. The container is filled up to no more than one third of its volume with a granular cultivation substrate, where the micro-organism grows. The heat generated by the growth of the micro-organism can be evaporated to a large extent by the partially cooled shell of the container. This happens during the cylinder""s slow rotation, which leads to the result, that the substrate comes again and again into contact with the shell and that it can evolve heat to it. However, the method has the disadvantage, that shear forces have an effect within the moving substrate, which lead especially to a destruction of fungal structures in development (mycelium, sporangium, fructovegetative body). In this way, it is, for example, for many fungi from the beginning not possible to obtain the goal of a high yield in spores. The problem of exsiccation is solved in this type of fermenter to a large extent by an aeration with moist air, because it is not necessary to evaporate the water from the substrate (evaporation coldness is not necessary). Moreover, spray nozzles, could achieve a moistening of the substrate, too providing a good distribution of free water by means of the movement.
However, large amounts of cultivation substrate lead to other problems in this type of fermenter:
1. The design of large fermenters is very costly.
2. The continuous movement of the fermenter can bring about an agglomeration of the moist substrate.
3. Interfaces to the exterior are necessary (air inlet and air outlet, water supply), which could easily become sources of foreign contamination by the rotation of the fermenter.
A similar fermenter as the xe2x80x98rotary drum bioreactorxe2x80x99 is the xe2x80x98swing solid state bioreactorxe2x80x99, with the only difference that the mixing of the substrate is not caused by rotary movement here, but by a shaking movement. Otherwise, the same, already mentioned advantages and disadvantages apply. An additional limitation of the volume for this type of fermenter is, however, applies, because the construction of the complicated shaking mechanism would hardly allow a weight of more than 100 kg for the filled container.
The xe2x80x98stirred vessel bioreactorxe2x80x99 can be described as a closed tank with a stirrer moving within. The problems for the use of large amounts of substrate are inevitable for this type of reactor, as these amounts can no longer be moved evenly without causing destructions in the structure of the substrates.
The cultivation substrate for the micro-organisms is kept constantly in a fluidized bed in the xe2x80x98air solid fluidized bed bioreactorxe2x80x99, which makes a relatively large volume of the reactor room necessary. The necessary air for keeping the fluidized bed up is conducted in a circulation. The air must be kept with an exactly calculated moisture content. This procedure requires a lot of energy for keeping the fluidized bed up. It could be demonstrated in an AiF project already conducted (Bahr, d.; Menner, M.: BIOforum 18 (1995), 16-21), that the cultivation of yeast cells is possible in the fluidized bed.
However, this was only achieved on a relatively small scale and at with rather small yields in comparison to the submerged fermentation. A cultivation of filamentous fungi on large amounts of granular cultivation substrate (more than 100 kg per batch) over several weeks with this technique is only possible at high costs, which are out of court.
Other state-of-the-art fermenters are too small to gain with them an economically profitable amount of fungal spores (EP-A1-0 683 815 and FR 85.08555), or it is not possible to exclude for a sufficient fermenter capacity a contamination of the cultivation substrate with nucleus of crystallization over a longer period of time (DE 4406632 C1).
Thus, the task for the present invention was to develop a SSF fermenter for large volumes and to provide a procedure for solid-state fermentation, which allows an economic application of the SSF of little competitive microorganisms in large fermenters.
It has to:
1. avoid a foreign contamination of the fermenter (keeping up of sterile conditions during the entire process of fermentation),
2. evacuate the heat, which is caused by the fungal metabolism without an exsiccation of the substrate (by increased air flow and use of the evaporation coldness),
3. avoid the occurrence of shear forces in the fermenter (no movement of the cultivation substrate) and
4. guarantee an even aeration (avoiding the exsiccation) and control over the temperature of the substrate.